Pharmaceutical composition for preventing and treating cancer and treating an inflammation

ABSTRACT

The present invention relates to a pharmaceutical composition preventing cancer and treating cancer and inflammation, which is characterized in that including xanthorrhizol as an active principle. Xanthorrhizol not only inhibits mutagenesis and tumor formation, and enhances the activity of detoxification enzyme of carcinogen, induces apoptosis of cancer cell, and suppresses the activity of COX-2 and iNOS which are related to tumor promotion and inflammatory reaction. Thus, a pharmaceutical composition including xanthorrhizol can be utilized for prevention of cancer and treatment of cancer and inflammation.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition forpreventing and treating cancer and treating an inflammation, moreparticularly, which not only inhibits generation of mutation and tumor,and enhances the activity of detoxification enzyme of carcinogen, andinduces apoptosis of cancer cell, but also suppresses the activity ofcyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS)enzyme which are related to the inflammatory reaction.

BACKGROUND ART

Cancer is now a major worldwide disease which causes 7 million people todie every year, and it was reported that more than about 1.5 millionpeople become new patients suffering from cancer in the United Satesannually in 1997. Considering this tendency, the cancer is assumed tobecome a leading cause of death before long.

It is known that cancer is caused by various factors. Carcinogens inducemutations by forming adducts to DNA or by bringing about damage to thegene, and it is well-known fact that mutation is a major factor ofcancer. Carcinogens are finally converted into ultimate carcinogens bymetabolism in the body as well as they flow directly into body.

Carcinogenesis can be classified into the three stages, i.e.,initiation, promotion and progression. Initiation begins when DNA in acell or population of cells is damaged by exposure to exogenous orendogenous carcinogens. If this damage is not repaired, it can lead togenetic mutations. The responsiveness of the mutated cells to theirmicroenvironment can be altered and may give them a growth advantagesrelative to normal cells. Promotion stage is characterized by selectiveclonal expansion of the initiated cells, a result of the alteredexpression of genes whose products are associated withhyperproliferation, tissue remodeling, and inflammation. During tumorprogression, preneoplastic cells (benign tumors) develop into malignanttumors through a process of clonal expansion that is facilitated byprogressive genomic instability and altered gene expression.

If benign tumors are progressed to malignant tumors, it is irremediable.Therefore, the recent studies are focused on preventing induction,inhibiting or delaying progression of cancers.

Many treatment methods, such as chemotherapy, radiotherapy, surgerytherapy and gene therapy, for curing cancer were developed. Among them,chemotherapy by medicine is most commonly used. In former days, theresearches to develop the synthetic anti-cancer drugs were performed,but recently, great concerns are concentrated on developing naturalmaterials that are useful for prevention and treatment of cancer.

To develop cancer chemopreventive agents inhibiting tumor formation,National Cancer Institute (NCI) in United State has announced 16compounds possessing chemopreventive potentials for clinical testreferred to Table 1. TABLE 1 Clinical test Preclinical test Phase IPhase II Phase III 1^(st)Generation Retinoids + + Vitamin A + + +13-cis-retinoic acid + + + + 4-HPR + + + Calcium + + + β-Carotene + + +Tamoxifen + Finasteride + + 2^(nd)Generation DFMO + + + Sulindac + +Piroxicam + + Oltipratz + + N-acetylcysteine + + Aspirin + +Ibuprofen + + Carbenoxole + + 18-β-Glycyrrhetinic acid + + DFMO +Piroxicam + + 3^(rd)Generation S-Allylcysteine + + Phenhexylisothiocyanate + Curcumin + Ellagic acid + Fumaric acid + Fluasterone +4-HPR + Oltipratz + 4-HPR + Tamoxifen +

Among the materials shown at Table 1, curcumin is a pigment componentseparated from Curcuma longa Linn: (Zingiberaceae) used as a traditionalfolk medicine in India. It is known that it has excellent anti-oxidanteffect and anti-inflammatory effect (Elizabath K. and Rao M. N. A., Int.J. Pharm., 58:237-240, 1990; Tonnesan H. H., Int. J. Pharm., 51:179-181, 1989), and excellent antimutagenic effect and anticarcinogeniceffect and the inhibitory effect on cell proliferation (Nagabhushan M.and Bhide S. V., J. Nutr. Growth Cancer, 4:83-89, 1987; Huang M. T., etal., Cancer Res., 48:5941-5946, 1988; Soudamini K. K. and Kuttan R., J.Ethnopharmacol., 27:227-233, 1989; Jee S. H., et al, J. Invest.Dermatol., 111, 656-661, 1998). Furthermore, it was reported thatcurcumin suppresses the tumor promotion induced by phobol ester, andshows cytotoxicity against cell lines of human leukemia, colon cancer,CNS, melanoma, kidney cancer and breast cancer (Ramsewak R. S., et al.,Phytomedicine, 7:303-308, 2000). NCI has planned a clinical test todevelop curcumin to chemopreventive agent (Kelloff G. J., et al., CancerEpidemiol. Biomarkers Prev., 3:85-98,1994).

Thus, natural products which not only show no side effects and inhibittumor formation and progression into malignant cancer but also cureinflammation closely related to tumor promotion are continuously beingdetected.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a pharmaceuticalcomposition not only preventing tumor formation but also treatingmalignant tumor (cancer) and inflammation by inhibiting mutagenesis andtumor formation by carcinogen, enhancing the activity of enzymes todetoxify carcinogen, inducing apoptosis of cancer cell, and suppressingthe activity or expression of COX-2 and iNOS which are closely relatedto tumor promotion and inflammation.

To achieve the object above-mentioned, the present invention provides apharmaceutical composition including xanthorrhizol as an effectivecomponent for preventing cancer and treating cancer and inflammation.

Xanthorrhizol is a sesquiterpenoid firstly separated from Curcumaxanthorrhiza by Rimpler et al. in 1970, which has a following chemicalstructure 1.

It is reported that xanthorrhizol suppresses the rigid shrinkage of thewomb of rat concentration-dependently (Ponce-Monter H., et al.,Phytother. Res., 13:202-205, 1999), and shows anti-bacterial activityagainst oral microorganisms such as Streptococcus mutans (Hwang J. K.,Fitoterapia, 71:321-323, 2000; Hwang J. K., Planta Med., 66:196-197,2000). Said xanthorrhizol could be extracted from Curcuma xanthoffhizaRoxb., a plant of Zingiberaceae family used as an Indonesian folkmedicine, and the extraction method such as extraction by organicsolvent, extraction by super-critical fluid, microwave extraction andultrasonic extraction can be used, as disclosed at Korean Patent LaidOpen No.2000-73295 and WO 88/05304.

We, the inventors have observed the inhibitory effects of xanthorrhizolon mutagenesis, tumor formation and inflammation. Xanthorrhizol enhancedthe activity of carcinogen-detoxifying enzyme, induced apoptosis ofcancer cell, inhibited the activity or expression of COX-2 and iNOSwhich is related to inflammation reaction. Therefore, our resultsindicate that xanthorrhizol could be effectively used for preventingcancer and treating cancer and inflammation.

The details of the efficacies of preventing cancer and treating cancerand inflammation of xanthorrhizol will be described as follows.

Most of carcinogens are mutagens. Tert-butylhydroperoxide or hydrogenperoxide is known as oxidative mutating agent which result in DNA damageand mutation by generating oxygen radical (Taffe B. G., et al., J. Biol.Chem., 262:12143-12149, 1987; Kappus H., Arch. Toxicol., 60:144-149,1987), particularly, tert-butylhydroperoxide acts as tumor-promotingagent on mouse skin by forming reactive oxygen species underphysiological condition (Epe B., et al., Environ. Health Perspect.,88:111-115, 1990). In the experiments of the present inventors,xanthorrhizol inhibits bacterial mutagenesis induced bytert-butylhydroperoxide or hydrogen peroxide more effectively thancurcumin.

Xanthorrhizol effectively inhibits tumor formation in two-stage mouseskin carcinogenesis model (DiGiovanni J., Pharmacol. Ther., 54:63-128,1992). It suggests that xanthorrhizol is a useful cancer chemopreventiveand anticarcinogenic agent.

In addition, xanthorrhizol induces the activation of Phase IIdetoxification enzyme which suppresses the tumor formation bydetoxifying carcinogens in the body. Xanthorrhizol can enhance theability of body detoxifying carcinogens by activatingQR[(NADP(H):quinone oxidoreductase)], a kind of Phase II detoxificationenzyme (Talalay P., et al., In: Cancer Biology and Therapeutics. eds. J.G. Cory and A. Szentivanyi. Plenum Press, New York, N.Y., pp. 197-216,1981). As a result, xanthorrhizol can control the early stage of tumorformation and tumor progression.

The activation of NF-κB increases in tumorigenesis (reference toCogswell P. C., et al., Oncogene, 19:1123-1131, 2000). The activation ofNF-κB is recognized to be critical for regulating the induction of COX-2and iNOS. One of the critical events in NF-κB activation is dissociationwith subsequent degradation of the inhibitory protein IκB viaphosphorylation and ubiquitination. Xanthorrhizol can effectivelyinhibit activation of NF-κB by suppressing degradation of IkBα. It couldbe understood from above result that xanthorrhizol is a useful agent toinhibit tumor formation.

Xanthorrhizol induces apoptosis of cancer cell. In the process ofapoptosis, it is known that the caspase called as interleukin-1βconverting enzyme (ICE) plays an important role [Martin, S. J. andGreen, D. R., Cell, 82:349-352, 1995]. The caspase group consists of atleast 10 caspase enzymes, and has subgroups of ICE(caspase-1,4,5),Ich-1(caspase-2,9), CPP32(caspase-3,6,7,8,10). If the procaspase isactivated to a caspase, it activates another caspase which is on thenext step, and poly(ADP-ribose)polymerase(PRAP)), a DNA repair enzyme,is decomposed by caspase-3 and activates DNA fragmentation-promotingfactor (DFF) to induce apoptosis [Liu X. S., et al., Cell, 89:175-184,1997]. Morphological characteristics such as DNA fragmentation andnuclear condensation observed commonly at the time of apoptosis show incancer cells treated with xanthorrhizol.

Xanthorrhizol could be effectively utilized for treatment ofinflammation by inhibiting expression of COX-2 and iNOS. It is knownthat the further each steps of tumorigenesis progresses, the more COX-2(cycleoxygenase-2) and iNOS (inducible nitric oxide synthase) expressionincrease (Kitayama W., et al., Carcinogenesis, 20:2305-2310, 1999;Takahashi M., et al., Cancer Res., 57:1233-1237, 1997). Accordingly, itcould be understood that there's a close relationship betweentumorigenesis and the inflammatory reaction.

Cyclooxygenase (COX) is a key enzyme that catalyzes the biosynthesis ofprostaglandins (PGs) from arachidonic acid. Two isoforms of COX,designated COX-1 and COX-2, have been identified. COX-1 isconstitutively expressed in most tissues and seems to be responsible forhousekeeping roles in normal physiological functions (Amiram R.,J.Biol.Chem., 263:3022-2024, 1988). In contrast, COX-2 is not detectiblein most normal tissues, but is induced by proinflammatory cytokines,growth factors, oncogenes, carcinogens, and tumor promoters, implying arole for COX-2 in both inflammation and control of cell growth(Subbaramaiah K., Cancer Res., 56:4424-4429, 1996). The increased levelof PGs in tumors is due, at least in part, to increased expression ofCOX-2. Overexpression of COX-2 also inhibits apoptosis and increases theinvasiveness of malignant cells (Tsujii M., et al.,Proc.Natl.Acad.Sci.USA, 94:3336-3340, 1997). Accordingly, compounds thatinhibit selectively the activity or expression of COX-2 might be animportant focus for cancer chemoprevention or anti-inflammation.

Nitric oxide synthase (NOS) is another important enzyme involved inregulation of inflammation, vascular tone, neurotransmission, tumorcells and other homeostasis of human body. NOS also exists in the twoforms of constitutive form and inducible form. The excessive generationof nitric oxide (NO) is related with pathological vasodilation,cytotoxicity and tissue injury. According to the recent results, NOSincreases the permeability of a blood vessel, causes inflammatoryreaction such as edema, and promotes the activation of COX to stimulatethe biosynthesis of inflammatory mediator such as prostaglandin toinduce severe inflammatory reaction. In various cancer tissue, theactivation of iNOS is highly increased. Therefore, xanthorrhizol whichsignificantly inhibits the activity of COX-2 and INOS could be utilizednot only for prevention of cancer, but also for treatment ofinflammation and cancer.

Pharmaceutical composition of the present invention includingxanthorrhizol preventing cancer and treating cancer and inflammationcould further comprise a pharmaceutically permissible vector and adiluent. Solvent, dispersion medium, absorption retardant and the likewhich are commercially used in the field of medicine industry can beused as a vector.

Pharmaceutical composition of the present invention for preventingcancer and treating cancer and inflammation could be dosed throughwhatever general route to reach the target tissue. Therefore, thecomposition of the present invention could be dosed through an affectedpart of the body, oral administration, parenteral administration,intra-narial cabity, intravenous injection, intramuscular injection,subcutaneous injection and intrascleral administration. The compositioncould be formulated as solution, suspended solution, tablet, pill,capsule and sustained releasing agent. The preferred formulation is aninjection, and the dosage content of the composition should bedetermined in consideration of the skill in the art according to thekinds and degree of disease, age, sex and so forth.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a graph representing the inhibitory effect of xanthorrhizol onbacterial mutagenesis induced by tert-butylhydroperoxide(a) and hydrogenperoxide(b).

FIG. 2 is a photograph of agar plate representing the inhibitory effectof xanthorrhizol on mutagenesis induced by hydrogen peroxide.

FIG. 3 is a graph representing the inhibitory effect of the methanolextract of Curcuma xanthorrhiza Roxb(A) and xanthorrhizol(B) againstskin tumor formation in two-stage mouse skin carcinogenesis induced by7,12-dimethylbenz[a]anthracene (DMBA) and12-O-tetradecanoyl-phorbol-13-acetate (TPA).

FIG. 4 is a photograph of mice showing the inhibitory effect ofxanthorrhizol against skin tumor formation in two-stage mouse skincarcinogeneis induced by DMBA and TPA.

FIG. 5 is a graph representing the increase of quinone reductase(QR)activity induced by xanthorrhizol.

FIG. 6 is a western blotting photograph representing that xanthorrhizolinhibits expression of COX-2 protein induced by TPA.

FIG. 7 is a graph representing the inhibitory effect of xanthorrhizol onlipopolysaccharide(LPS)-activated PGE2 production(COX-2 activity).

FIG. 8 is a graph representing the inhibitory effect of xanthorrhizol onLPS-activated nitric oxide production (iNOS activity).

FIG. 9 is a western blotting photograph representing the inhibitoryeffect of xanthorrhizol on decomposition of IkBα.

FIG. 10 is an agarose gel photograph representing DNA fragmentationinduced by xanthorrhizol.

FIG. 11 is a flow cytometric analysis representing the induction ofapoptosis by xanthorrhizol.

FIG. 12 is a western blotting photograph representing the activation ofprocaspase-3 by xanthorrhizol.

EMBODIMENTS

The more detail description of the present invention is best understoodwith reference to the preferred embodiments. But the preferredembodiments of the present invention can be variously modified, and therange of the present invention should not be limited to the followingembodiments. The embodiments of the present invention are provided forillustrating the present invention more completely to those skilled inthe art.

The experimental result is represented as an mean ±SE and IC₅₀, and IC₅₀is the concentration inhibiting 50% of the reaction. Difference betweenmeans of various subgroups is assessed by Student t-test. Statisticalsignificance is defined as a value of P<0.05.

Example of Separation and Purification of Xanthorrhizol

After extracting the dried rhizome of Curcuma xanthorrhiza with 75%methanol, the extract was fractionated with ethylacetate, butanol,water. A certain single material was purified from ethylacetate fractionby silica gel column chromatography eluted with the mixture ofhexane/ethylacetate (10:1, v/v). The purified material was determined tobe xanthorrhizol by measuring the molecular weight using EI-MS and byanalyzing the ¹H-NMR, ¹³C-NMR and IR spectrum of it.

IR(CDCl₃, V_(max)) 3402, 2915, 1708, 1620, 1599 cm⁻¹;

EI-MS(m/z) 218, 148, 136, 135, 121; ¹H-NMR(CDCl₃, 400 MHz) 1.18(3H, d,J=7.1 Hz), 1.52(3H, s), 1.57(2H, dt, J=7.1, 7.2 Hz), 1.67 (3H, s),1.85(2H, dt, J=7.0, 7.2 Hz), 2.20(3H, s), 2.59(1H, qt), 5.08(1H, t,J=7.0, 7.2 Hz), 6.59(1H, br s), 6.66(1H, br d), 7.01(1 H, d, J=7.6 Hz);

¹³C-NMR(CDCl₃, 400 MHz) 147.16, 113.50, 153.51, 120.86, 130.74, 119.42,38.98, 38.32, 26.10, 124.48, 131.39, 15.31, 25.67, 17.64, 22.3

Embodiment 1 The Antimutagenic Effect on Mutagenesis Induced by ReactiveOxygen Species

The antimutagenic effect of xanthorrhizol was examined in Salmonellatyphimurium TA102 strain including mutagenesis with reactive oxygenspecies (Levin, D. E., et al., Proc. Natl. Acad. Sci. U. S. A.,79;7445-7449, 1982).

Salmonella typhimurium TA102 strain was cultured in Oxoid nutrient brothmedium for 11 hours. 100 μl of above-cultured medium was added to 600 μlof the reaction mixture containing tert-butylhydroperoxide (100μg/plate) or hydrogen peroxide (50 μg/plate) with or withoutxanthorrhizol and incubated for 30 minutes at 37° C. Curcumin was addedinstead of xanthorrhizol in positive control. The concentration ofxanthorrhizol or curcumin was 0, 10, 20, 40, 60 nmol/plate and 2, 4, 8,10, 20, 50 nmol/plate respectively in experiment to examine theinhibitory effect of xanthorrhizol against tert-butylhydroperoxide andhydrogen peroxide-induced mutagenesis. The reaction mixture wastransferred to 2 ml of top agar solution containing 0.5 mM of histidineand biotin and was homogeneously mixed. It was poured to minimal glucoseplate. The plates were incubated for 48 hours at 37° C. and the numberof His+ revertant colonies counted.

The antimutagenic effect against mutagenesis induced bytert-butylhydroperoxide(a) and hydrogen peroxide(b) was represented atgraph (A) and (B) at FIG. 1, respectively, and a photograph of agarplate representing the antimutagenic effect of xanthorrhizol againstmutagenesis induced by hydrogen peroxide are shown at FIG. 2. As shownin FIG. 2, xanthorrhizol showed more excellent inhibitory effect againstmutagenesis induced by tert-butylhydroperoxide and hydrogen peroxidethan curcumin used as a positive control.

Embodiment 2 The Inhibitory Effect on Tumor Formation in Two-Stage MouseSkin Carcinogenesis Model

The chemoprotective effect of xanthorrhizol and the methanolic extractof Curcuma xanthorrhiza Roxb. against tumor formation was investigatedin multistage mouse carcinogenesis induced by tumor initiator (DMBA) andtumor promoter (TPA).

The methanolic extract of Curcuma xanthorrhiza Roxb. was prepared asfollows. After cutting the dried Curcuma xanthorrhiza into small pieces,400 ml of 75% methanol was added to 100 g of the sample and extractedrepeatedly for 2 days at room temperature. The methanolic extract wasfiltered with Whatman filter paper, evaporated and dried byfreeze-drier.

To evaluate the inhibitory effect of xanthorrhizol and the methanolicextract of Curcuma xanthoffhiza Roxb. against tumor formation, 30 mice(6 weeks age, female) per an experimental group was used. The dorsalregion of ICR mice was shaved with an electric clipper. After a topicalapplication of 0.2 μmol DMBA in 0.2 ml acetone, mice were treatedtopically with xanthorrhizol or the methanolic extract of Curcumaxanthorrhiza 30 min prior to each topical application of 10 nmol TPA in0.2 ml acetone which was continued three times weekly for 19 weeks. Thenegative control was treated with only 0.2 ml acetone. Tumors werecounted and recorded biweekly. The results were expressed as the averagenumber of tumors per mouse (tumor multiplicity) and the percentage oftumor-bearing mice (tumor incidence) and are shown at FIG. 3 and FIG. 4.The graph (A) of FIG. 3 represents the tumor multiplicity of eachexperimental group and the graph (B) shows the tumor incidence. FIG. 4is a photograph representing the inhibitory effect of xanthorrhizolagainst tumor formation at 19 weeks.

As shown in FIG. 3 and FIG. 4, xanthorrhizol inhibits tumor formationdose-dependently. All of the mice treated with DMBA and TPA withoutxanthorrhizol had tumors with an average of 15.5 skin tumors. On theother hand, mice given topical application of 6 μmol xanthorrhizol threetimes per week for 19 weeks developed an average of 4.0 skin tumors permouse and 57% of the treated mice had tumors. These results indicatethat xanthorrhizol is an excellent chemopreventive agent reducing tumorincidence and tumor multiplicity significantly.

Embodiment 3 Induction of Quinone Reductase Activity

Hepa 1c1c7 cell (2.5×10⁴/ml), a liver cancer cell of rat, was seededinto 96 well plate and was cultured in 10% FBS-αL-MEM (Gibco BRL) at 37°C. for 24 hours in 5% CO₂ of humidified air. 190 μl of fresh media and10 μl of xanthorrhizol dissolved in 10% of DMSO was added to aboveculture media and it was cultured under 5% CO₂ at 37° C. for 48 hours.The culture media were discarded, and after washing with PBS (phosphatebuffered saline), 50 μl of reaction solution containing 0.8% digitoninand 2 mM EDTA was added to each well, and it was cultured for 10 minutesto destroy the cell. After the plate was shaken in the orbital shaker(100 rpm) for 10 minutes, 200 μl of reaction solution containingmenadione and MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide) (final reaction solution 50 ml: 2.5 mL of 0.5M-tris, 0.34 ml of1.5% Tween-20, 0.034 ml of 7.5 mM FAD, 0.334 ml of 150 mM G-6-P, 30 μlof 50 mM NADP, 100 μl of Glucose 6-phosphate dehydrogenase, 33.4 mg ofBSA, 15 mg of MTT, 50 μl of 50 mM menadione) was added to react for 10minutes. Then, 50 μl of 5 mM potassium phosphate (pH 7.4) solutioncontaining 0.3 mM of dicoumarol was added to terminate the reaction andthe absorbance at 595 nm was measured spectrophotometrically.

To assess the effect of xanthorrhizol on cell growth, protein wasmeasured in the 2^(nd) plate set cultured under same condition above.After removing the culture media, the cell was treated with 0.2% crystalviolet for 10 minutes, then washed with tap water and dried. And 200 μlof 0.5% SDS was added to cell and mixed, then the absorbance at 595 nmwas measured spectophotometrically.

To estimate the experimental result, firstly, QR specific activity ofeach group treated with xanthorrhizol and the control group wascalculated by following equation 1. The relative level of QR activityinduced by xanthorrhizol, that is, QR induction ratio (treated/control)was defined as the ratio between QR specific activity of the grouptreated with xanthorrhizol and that of control by following equation 2.The concentrations of xanthorrhizol used were 50, 10, 2, 0.4 μM,respectively.QR specific activity=(Absorbance change of MTT per min/Absorbance changeof crystal violet)×3247 nmol/mg   [Equation 1]QR induction ratio=Specific activity of test sample treated withxanthorrhizol/Specific activity of control   [Equation 2]

QR induction ratio by xanthorrhizol represented at FIG. 5. As shown inFIG. 5, QR induction ratio at 0.4 μM and 50 μM of xanthorrhizol is about125% and 130%, respectively, compared with the control. These resultssuggest that xanthorrhizol could contribute to removal of carcinogen inthe body by increasing the activity of enzyme detoxifying carcinogenssuch as QR.

Embodiment 4 Inhibition of COX-2 Expression Induced by TPA

It is known that the expression of COX-2 increases in mouse skin treatedwith TPA. Therefore, the effect of xanthorrhizol on COX-2 expressioninduced by TPA was measured as follows on the basis of this fact.

Female ICR mice of about 5 weeks of age were purchased from the DaehanExperimental Animal Center (Seoul, Korea). Mice were kept on a 12 hlight/dark cycle.

The dorsal region of mice was shaved with an electric clipper. 2 dayslater, xanthorrhizol dissolved in 0.2 ml acetone was topically appliedon mouse skin followed by topical application of TPA (10 nmol) dissolvedin 0.2 ml acetone after 30 min. Mice were sacrificed by cervicaldislocation 4 hr later. The skin was excised and the fat was removed.Fat-free skin was immediately placed in liquid nitrogen and pulverizedin mortar.

Pulverized mouse skin was lysed in 400 μl of lysis buffer [150 mM NaCl,0.5% Triton X-100, 50 mM tris-HCl, pH 7.4, 20 mM EGTA, 1 mM DTT, 1 mMNa₃VO₄, protease inhibitor cocktail tablet] for 30 min on ice. Lysateswere centrifuged and total protein in supernatant was quantified byBio-Rad protein assay. Aliquots of supernatant containing 30 μg proteinwere boiled in SDS sample loading buffer for 5 min beforeelectrophoresis on a 12% SDS-polyacrylamide gel. Blots were transferredfrom SDA-polyacrylamide gel to PVDF membrane, blocked with 5% fat-freedry milk-PBS buffer containing 0.1% Tween 20 (PBST) for 2 hr at roomtemperature and then washed with PBST buffer. Membranes were incubatedfor 1 hr at room temperature with goat COX-2 polyclonal antibody for 2hr. Blots were rinsed with PBST, incubated with anti-goat horseradishperoxidase-conjugated secondary antibody (Zymed Laboratories Inc., SanFrancisco, Calif., USA) and then washed again 3 times in PBST buffer for5 min. Transferred proteins were visualized with an ECL (Enhancedchemiluminescence) detection kit. Western blotting of COX-2 was shown inFIG. 6. Referring FIG. 6, the expression of COX-2 induced by TPA wasdecreased by pretreatment with xanthorrhizol in a dose-dependent manner.

Embodiment 5 Inhibition of COX-2 Activity Induced by Lipopolysaccharide(LPS)

If a cell was treated with LPS, the activity of COX-2 increases. On thebasis of this fact, to investigate the effect of xanthorrhizol onLPS-induced COX-2 activity, the quantity of PGE₂ released from cells wasmeasured as follows.

RAW264.7 macrophage cells were maintained in DMEM supplemented withpenicillin-streptomycin and 10% FBS at 37° C., in 5% CO₂ of humidifiedair. The cells (10×10⁵ cells/ml, 200 μl) were allowed to adhere for 4 hrin the presence of aspirin (500 μM) in a 96-well culture plate toinhibit irreversibly COX activity in cells, washed 3 times with media,and then incubated in the fresh medium with 1 μg/ml of LPS.Xanthorrhizol was simultaneously added to each well. After an additional16 hr incubation, the media were recovered and analyzed by PGE₂ enzymeimmunometric assay. The medium recovered from each well was added toeach well attached anti-PGE₂ antibody (Amersham Life Science, ArlingtonHeights, Ill.) with PGE₂-acetylcholineesterase tracer, incubated for 18hr at room temperature and then washed five times with 0.05% Tween20-phosphate buffer solution. 200 μl of Ellman reagent was added to eachwell and incubated for 7 hr. Absorbance at 405 nm was measured. PGE₂ ineach medium treated with xanthorrhizol was quantified in calibrationcurve graphed with standard PGE₂. 100% activity is defined as thedifference between PGE₂ accumulation in the absence and in the presenceof LPS for 16 hr in triplicate determinations. The percentage inhibitionwas expressed as [1-(PGE₂ level of sample/PGE₂ level of vehicletreated−control)]×100. The result is shown in FIG. 7.

FIG. 7 demonstrates that xanthorrhizol inhibits the activity of COX-2induced by LPS dose-dependently, especially xanthorrhizol shows not lessthan 98% of percentage inhibition (IC₅₀=0.07 μg/ml=0.32 μM) at theconcentration of not less than 1 μg/ml. This result suggests thatxanthorrhizol can inhibit inflammation and tumor promotion by blockingCOX-2 activity.

Embodiment 6 Inhibition of iNOS Activity Induced by LPS

The effect of xanthorrhizol on iNOS activity induced by LPS wasmeasured. RAW264.7 macrophage cells were maintained in DMEM supplementedwith penicillin-streptomycin and 10% FBS at 37° C., in 5% CO₂ ofhumidified air. The cells in 10% FBS-DMEM without phenol red media wereplated in 24-well plates (8×10⁵/ml), and then incubated for 4 hr. Thecells were replaced with new media, and incubated in the medium with 1μg/ml of LPS and xanthorrhizol. After an additional 20 hr incubation,the media were removed and analyzed for nitrite accumulation as anindicator of NO production by the Griess reaction. 150 μl of Griessreagent were added to 100 μl of each supernatant from LPS and/orxanthorrhizol treated cells in triplicate. The plates were incubated for10 min, and were read at 570 nm against a standard curve of NaNO₂. Thepercentage inhibition was expressed as [1−(NO level of sample/NO levelof vehicle treated−control)]×100. The result is shown in FIG. 8.

Referring to FIG. 8, xanthorrhizol inhibits dose-dependently theactivity of iNOS induced by LPS, and particularly shows not less than99% of percentage inhibition at the concentration of 10 μg/ml (IC₅₀=1.01μg/ml=4.63 μM). This result suggests that xanthorrhizol can mitigateinflammation and tumor promotion by inhibiting production of nitricoxide.

Embodiment 7 Inhibition of lκB Degradation in Mouse Skin Treated withTPA

To examine the effect of xanthorrhizol on IκB, the level of IκB wasmeasured in mouse skin. Cytoplasmic extract was prepared as follows. Themouse skin tissue obtained by the same method of embodiment 4 washomogenized in hypotonic buffer solution [10 mM HEPES, pH 7.8, 10 mMKCl, 2 mM MgCl₂, 1 mM DTT, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF)]. To the homogenates was added 125 μl of 10% NondietP-40 solution and the mixture was then centrifuged for 30 sec. Thesupernatant (cytoplasmic extract) was electrophoresized on the 12%SDS-polyacrylamide gel. Blot was transferred from SDS-polyacrylamide gelto PVDF membrane, blocked with 5% fat-free dry milk-PBST buffer for 2 hrat room temperature and then washed in PBST buffer. Membrane wasincubated for 2 hr at room temperature with rabbit IκBα polyclonalantibody (Santa Cruz Product, Santa Cruz, Calif., USA). Blot was rinsedwith PBST, incubated with anti-rabbit horseradish peroxidase-conjugatedsecondary antibody (Santa Crus product, Santa Cruz, Calif., USA) andagain washed 3 times in PBST buffer for 5 min. Transferred protein wasvisualized with an ECL detection kit. The western blotting photographwas shown in FIG. 9. Referring FIG. 9, it could be understood that thedegradation of IκBα induced by TPA is inhibited by xanthorrhizol in adose dependent manner.

Embodiment 8 Induction of Apoptosis by Xanthorrhizol

Human promyelocytic leukemia (HL-60) cells were maintained at 37° C. ina humidified atmosphere of 95% air and 5% CO₂ in RPMI 1640 supplementedwith 10% (v/v) heat-inactivated fetal bovine serum (FBS). HL-60 cellswere cultured in 6-well plate in RPMI 1640 medium containing 10% FBS inthe absence or presence of the methanolic extract of Curcumaxanthorrhiza (15 μg/ml) and xanthorrhizol (40 μM) and centrifuged after24 hr. 4% neutral buffered formaline was added to the cell and themixture was transferred to slides, which were left at room temperaturefor dryness. The fixed cells were washed in PBS, air-dried and stainedwith DNA-specific fluorochrome Hoechest 33258 for 1 min. The adheredcells were washed with PBS, air dried, and mounted with 50% glycerol.The slides were observed by fluorescence microscopy. The result showedmorphological characteristics of apoptosis such as distinct chromatincondensation and nuclear fragmentation in HL-60 cells treated by Curcumaxanthorrhiza and xanthorrhizol.

HL-60 cells were cultured in 10% FBS-RPMI 1640 medium of 100 mm Petridish for 2 days. The cells were treated with 0,10, 40, 80 μM ofxanthorrhizol to investigate the effect of xanthorrhizol on DNAfragmentation, a biochemical marker of apoptosis. After 24 hr, the cellswere collected, incubated with 500 μl of lysis buffer (1 % Triton-X 100,50 mM Tris-HCl pH 7.4, 20 mM EDTA) for 1 hr on ice, and centrifuged. Tothe supernatant was added 100 μl of 1% SDS, 10 μl f TE/RNase(10 mg/ml),50 pl of proteinase K (1 mg/ml) and the mixture was incubated at 37° C.at least for 4 hr. DNA was extracted withphenol-chloroform-isoamylalcohol (25:24:1, v/v) and precipitated at −70°C. for 1 hr after addition of 2.5 volumes of cold ethanol. DNA fragmentswere resolved by 1.5% agarose gel electrophoresis and visualized bystaining with ethidium bromide. The result of electrophoresis is shownin FIG. 10, demonstrating that DNA fragmentation, a biochemical markerof apoptosis, was induced by 80 μM of xanthorrhizol.

The effect of xanthorrhizol on cell cycle was examined by flowcytometric analysis. HL-60 cells were cultured in serum-free RPMI 1640medium for 48 hr to stop cell cycle at GO phase. The medium wasexchanged to 10% FBS-RPMI 1640 media with 0, 20, 60 μM of xanthorrhizol,respectively. 24 hours later, the cells obtained after centrifugationwere fixed in 70% ethanol at −20° C. overnight. The cells were washedtwice again with PBS, and incubated with 100 U/ml of Rnase at 37° C. for1 hr. The cell pellet was resuspended in propidium iodide solution afterwashing twice with PBS. The cells were analyzed by flow cytometry andthe result was represented at FIG. 11.

As shown in FIG. 11, 20% in control and 36% and 76% in cells treatedwith 20 μM and 60 μM of xanthorrhizol respectively were the proportionsof cells in sub-G1 phase compartments [apoptosis peak, M1 fraction,sub-diploid DNA content]. This result shows that xanthorrhizol inducesapoptosis concentration-dependently.

Embodiment 9 Activation of Procaspase-3 by Xanthorrhizol

To investigate whether xanthorrhizol also induces the activation ofprocaspase-3, HL-60 cells were treated with 0, 10, 40, 80 μM ofxanthorrhizol for 24 hr and was also treated with 80 μM of xanthorrhizolfor 0, 2, 4, 6, 9 and 12 hr. The cells were harvested, suspended in 400μl of lysis buffer described in embodiment 4, incubated 4° C. for 40 minand centrifuged. The supernatant was electrophoresized on the 12%SDS-polyacrylamide gel. Blot was transferred from SDS-polyacrylamide gelto PVDF membrane, blocked with 5% fat-free dry milk-PBST buffer for 2 hrat room temperature and then washed in PBST buffer. Membrane wasincubated for 2 hr at room temperature with mouse procaspase-3monoclonal antibody (Transduction Laboratories, Lexington, Ky., USA).Blot was rinsed with PBST, incubated with mouse horseradishperoxidase-conjugated secondary antibody and again washed 3 times inPBST buffer for 5 min. Transferred protein was visualized with an ECLdetection kit. The western blotting photograph of procaspase-3 is shownin FIG. 12.

Referring FIG. 12, 40 μM of xanthorrhizol activated the procaspase-3 tocaspase-3.

Taken together, xanthorrhizol inhibits bacterial mutagenesis and mouseskin formation, enhances the activity of carcinogen-detoxifying enzyme,induces apoptosis of cancer cell and suppresses significantly theactivity and expression of COX-2 and iNOS which are closely related totumor promotion as well as inflammation. Therefore, a pharmaceuticalcomposition including xanthorrhizol is very useful for prevention ofcancer and treatment of cancer and inflammation.

1-2. (canceled)
 3. A method of treating or preventing cancer in a humanor animal which comprises administering to the human or animal in needthereof an effective amount of xanthorrhizol.
 4. A method of treating orpreventing inflammation in a human or animal comprising administering tothe human or animal in need thereof an effective amount ofxanthorrhizol.
 5. The method of claim 3 or 4, wherein the administrationis oral or parenteral.
 6. The method of claim 3 or 4, wherein theadministration is rectal, vaginal, topical or transdermal.
 7. The methodof claim 5, wherein the administration is intravenous, intramuscular,intraperitoneal, or subcutaneous.
 8. A pharmaceutical compositionsuitable for treating or preventing cancer or inflammation comprising aneffective amount of xanthorrhizol and a pharmaceutically acceptablecarrier, diluent or excipient.
 9. The pharmaceutical composition ofclaim 8, wherein the pharmaceutical composition is adapted for oral orparenteral administration to a patient.
 10. The pharmaceuticalcomposition of claim 8, wherein the pharmaceutical composition isadapted for rectal, vaginal, topical or transdermal administration to apatient.
 11. The pharmaceutical composition of claim 9, wherein theadministration is intravenous, intramuscular, intraperitoneal orsubcutaneous.
 12. A single unit dosage form which comprises thepharmaceutical composition of claim
 8. 13. The dosage form of claim 12wherein the dosage form is adapted for oral or parenteral administrationto a patient.
 14. The dosage form of claim 12 wherein the dosage form isadapted for rectal, vaginal, topical or transdermal administration to apatient.
 15. The pharmaceutical composition of claim 13, wherein theadministration is intravenous, intramuscular, intraperitoneal orsubcutaneous.